Glycosylation as a tracer of off-target Cre-lox activation in development.

The Cre-lox system is one of the most widely used methods for lineage-specific and inducible genome editing in vivo. However, incomplete penetrance and off-target effects due to transient promoter expression in a stem or pluripotent precursor cell can be problematic and difficult to detect, especially if the target gene is not normally present in the fully differentiated but off-target cells. Yet, the loss of the target gene through the transient expression of Cre may impact the differentiation of those cells by virtue of transient expression in a precursor population. In these situations, off-target effects in an unknown precursor cell can, at best, complicate conclusions drawn from the model, and at worst, invalidate all data generated from that knockout strain. Thus, identifying Cre-driver promoter expression along entire cell lineages is crucial to improve rigor and reproducibility. As an example, transient expression in an early precursor cell has been documented in a variety of Cre strains such as the Tie2-based Cre-driver system that is used as an "endothelial cell-specific" model 1. Yet, Tie2 is now known to be transiently expressed in a stem cell upstream of both hematopoietic and endothelial cell lineages. Here, we use the Tie2 Cre-driver strain to demonstrate that due to its ubiquitous nature, plasma membrane glycans are a useful marker of both penetrance and specificity of a Cre-based knockout.

Antigen presenting cell response to polysaccharide A is characterized by the generation of anti-inflammatory macrophages.

Polysaccharide A (PSA) is the immunodominant capsular carbohydrate from the gram negative commensal microbe Bacteroides fragilis that has shown remarkable potency in ameliorating many rodent models of inflammatory disease by eliciting downstream suppressive CD4+ T cells. PSA is composed of a zwitterionic repeating unit that allows it to be processed by antigen presenting cells (APCs) and presented by MHCII in a glycosylation-dependent manner. While previous work has uncovered much about the interactions between MHCII and PSA, as well as the downstream T cell response, little is known about how PSA affects the phenotype of MHCII+ APCs, including macrophages. Here, we utilized an unbiased systems approach consisting of RNAseq transcriptomics, high-throughput flow cytometry, Luminex analysis and targeted validation experiments to characterize the impact of PSA-mediated stimulation of splenic MHCII+ cells. The data revealed that PSA potently elicited the upregulation of an alternatively activated M2 macrophage transcriptomic and cell surface signature. Cell-type-specific validation experiments further demonstrated that PSA-exposed bone marrow-derived macrophages (BMDMs) induced cell surface and intracellular markers associated with M2 macrophages compared with conventional peptide ovalbumin (ova)-exposed BMDMs. In contrast to macrophages, we also found that CD11c+ dendritic cells (DCs) upregulated the pro-T cell activation costimulatory molecule CD86 following PSA stimulation. Consistent with the divergent BMDM and DC changes, PSA-exposed DCs elicited an antigen-experienced T cell phenotype in co-cultures, whereas macrophages did not. These findings collectively demonstrate that the PSA-induced immune response is characterized by both T cell stimulation via presentation by DCs, and a previously unrecognized anti-inflammatory polarization of macrophages.

IgG sialylation occurs via the FcRn-mediated recycling pathway in endothelial cells.

IgG is a key mediator of immune responses throughout the human body, and the structure of the conserved glycan on the Fc region has been identified as a key inflammatory switch regulating its downstream effects. In particular, the absence of terminal sialic acid has been shown to increase the affinity of IgG for activating Fc receptors, cascading the inflammatory response in a variety of diseases and conditions. Previously, we have shown that IgG sialylation is mediated by B cell-extrinsic processes. Here, we show that the FcRn-mediated recycling pathway within endothelial cells is a critical modulator of IgG sialylation. Building a deeper understanding of how IgG sialylation is regulated will drive the development of novel therapeutics which dynamically tune IgG functionality in vivo.

Acute and chronic lung inflammation drives changes in epithelial glycans.

Introduction: Asthma is the most common chronic inflammatory disease and it is characterized by leukocyte infiltration and tissue remodeling, with the latter generally referring to collagen deposition and epithelial hyperplasia. Changes in hyaluronin production have also been demonstrated, while mutations in fucosyltransferases reportedly limit asthmatic inflammation. Methods: Given the importance of glycans in cellular communication and to better characterize tissue glycosylation changes associated with asthma, we performed a comparative glycan analysis of normal and inflamed lungs from a selection of murine asthma models. Results: We found that among other changes, the most consistent was an increase in fucose-α1,3-N-acetylglucosamine (Fuc-α1,3-GlcNAc) and fucose-α1,2-galactose (Fuc-α1,2-Gal) motifs. Increases in terminal galactose and N-glycan branching were also seen in some cases, whereas no overall change in O-GalNAc glycans was observed. Increased Muc5AC was found in acute but not chronic models, and only the more human-like triple antigen model yielded increased sulfated galactose motifs. We also found that human A549 airway epithelial cells stimulated in culture showed similar increases in Fuc-α1,2-Gal, terminal galactose (Gal), and sulfated Gal, and this matched transcriptional upregulation of the α1,2-fucosyltransferase Fut2 and the α1,3-fucosyltransferases Fut4 and Fut7. Conclusions: These data suggest that airway epithelial cells directly respond to allergens by increasing glycan fucosylation, a known modification important for the recruitment of eosinophils and neutrophils.

Divergent Golgi trafficking limits B cell-mediated IgG sialylation.

The degree of α2,6-linked sialylation on IgG glycans is associated with a variety of inflammatory conditions and is thought to drive IgG anti-inflammatory activity. Previous findings revealed that ablation of ß-galactoside α2,6-sialyltransferase 1 (ST6Gal1) in B cells failed to alter IgG sialylation in vivo, yet resulted in the loss of B cell surface α2,6 sialylation, suggesting divergent pathways for IgG and cell surface glycoprotein glycosylation and trafficking. Employing both B cell hybridomas and ex vivo murine B cells, we discovered that IgG was poorly sialylated by ST6Gal1 and highly core fucosylated by α1,6-fucosyltransferase 8 (Fut8) in cell culture. In contrast, cell surface glycoproteins on IgG-producing cells showed the opposite pattern by flow cytometry, with high α2,6 sialylation and low α1,6 fucosylation. Paired studies further revealed that ex vivo B cell-produced IgG carried significantly less sialylation compared with IgG isolated from the plasma of matched animals, providing evidence that IgG sialylation increases after release in vivo. Finally, confocal analyses demonstrated that IgG poorly localized to subcellular compartments rich in sialylation and ST6Gal1, and strongly to regions rich in fucosylation and Fut8. These findings support a model in which IgG subcellular trafficking diverges from the canonical secretory pathway by promoting Fut8-mediated core fucosylation and limiting exposure to and modification by ST6Gal1, providing a mechanism for why B cell-expressed ST6Gal1 is dispensable for IgG sialylation in vivo.